Abstract [en]

This thesis focuses on protein structure, dynamics and interaction and their relation to human disease. In particular, the biophysical and structural properties of both well-ordered and partially disordered proteins are studied using a range of biophysical techniques such as circular dichroism spectroscopy, fluorescence spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. Pseudomonas aeruginosa is a human pathogen due to its multidrug resistance (MDR) caused by overexpression of efflux pump systems. This thesis describes how MDR mutations within the MexR repressor of the MexAB-OprM system reduce the DNA affinity by altering its stability with maintained structure. The oncogenic protein c-Myc is involved in many essential biological functions such as cell proliferation, differentiation and apoptosis and is also highly associated with several forms of human cancers, and where the N-terminal domain is regulated by a plethora of protein interactions. In this thesis the intrinsically disordered N-terminal part of c-Myc and its interactions with the proteins Bin1 and TBP are described. Myc binds Bin1 with maintained disorder in a multivalent manner, which may explain why the onco-protein can interact with such a wide range of binding partners. A similarly dynamic interaction is observed for Myc with the TATA-binding protein (TBP). The essential human multidomain glutaredoxin Grx3 is associated with several biological functions such as redox signaling, proliferation and signal transduction. We have solved the structure and analyzed the dynamic properties in the ps-ns and ms time scale for the two N-terminal domains, providing a platform for further analysis of the Grx3 protein and its interactions. Taken together, this thesis emphasizes the importance of joint structural, biophysical and dynamic studies to better understand protein function in health and disease.

Abstract [en]

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.

Abstract [en]

The crucial role of c-Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of c-Myc function. The transactivation domain of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of c-Myc-1-88, where hierarchical phosphorylation of T58 and S62 regulates activation and destruction of the c-Myc protein. By NMR chemical shift analysis, relaxation measurements and NOE analysis, we show that both the MBI region (residues 45-65) and residues 22-33 are transiently structured regions, conserved also in other members of the Myc family. Binding of Bin1-SH3 to c-Myc-1-88 as assayed by NMR and SPR revealed primary binding to the S62 region, but also a dynamically disordered and multivalent complex in which intrinsic disorder of c-Myc-1-88 was retained while releasing transient intramolecular interactions. Our findings describe a novel mode of regulatory recognition of c-Myc that is in agreement with the increasingly recognized capability of intrinsically disordered regions to efficiently mediate transient interactions with a wide range of targets, with important implications towards understanding the unique multifaceted biological functions of c-Myc.

Abstract [en]

The proto-oncogene c-myc affects the occurrence, expansion, and evolution of numerous aggressive human cancers, and is often associated with the late-stage and/or poor prognostic disease. Regulation of target gene activity by c-Myc occurs through protein interactions with the c-Myc transactivation domain (TAD) which, in addition to binding the TATA-binding protein (TBP) also recruits a wide variety of co-activators and suppressor proteins. Here, we present a molecular model, based on NMR, X-ray crystallography and SPR measurements, which describes how the c-Myc TAD binds to TBP. Our model contributes to the understanding of how c-Myc can regulate individual genes as well as entire gene programs.

Lundström, Patrik

Abstract [en]

Human glutaredoxin (Grx3) is an essential protein associated with biological functions including embryonic development and immune response, and is involved in human disease such as lung, colon cancer and cardiovascular disorder. Grx3 can harbour a [2Fe-2S]2+ cluster and is most likely involved in oxidative stress response. Grx3 consists of an N-terminal thioredoxin-like domain and two additional monothiol glutaredoxin domains, and is thus classified as a multidomain monothiol glutaredoxin. The Grx3 thioredoxin domain lacks both a characteristic active-site and catalytic activity, but is still essential in the yeast homologue and presumably functions together with its monothiol glutaredoxin domains. We have characterised the structures of the two Nterminal domains in Grx3, which have thioredoxin and glutaredoxin folds. We have analysed their dynamic and structural interdependence by analysing NMR relaxation data together with chemical shift changes between isolated and covalently linked domains. We find that although the two domains show interdomain mobility around a semi-flexible linker, there are indications for a preferred interaction surface between the two domains. Millisecond internal dynamics in a suggested ligand binding site in the isolated thioredoxin domain is dampened in the domain pair, suggesting that the two domains mutually affect each other on a profound level. Our results present a platform for further detailed studies of multidomain thioredoxin-glutaredoxin containing proteins, and their function in human cells.

Abstract [en]

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750–2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3′,5′-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.